Most of the studies of essential amino acid requirements with semi-synthetic
diets were conducted during the 1950s and 1960s using nitrogen
balance or nitrogen retention as the criterion of adequacy. Dietary
levels of a single amino acid were varied in successive periods of 5 to
10 days duration. These studies have been reviewed in detail by Irwin
and Hegsted (1). In addition to studies with varying quantities of
single amino acids, investigations with different amounts of complete
essential amino acid mixtures or single food proteins of known amino acid
composition and digestibility have also provided information and verification
of essential amino acid requirements. The results of all of these investigations
have been considered and evaluated by both the FAO/WHO Expert
Committee on Energy and Protein Requirements (2) and the Committee on
Amino Acids of the Food and Nutrition Board (3). In this paper new
information published since the deliberations of these committees will be
reviewed. Attention will be given to investigations of biochemical
indices that might prove useful in assessment of amino acid requirements
and factors which may affect amino acid requirements will be considered.

Amino Acid Requirements of Infants and Children

The experimental data on amino acid requirements for infants and
10–12-year-old children have been expanded by Pineda and co-workers (4) to
include requirements for the 2-year-old child. Forty-two healthy children
received diets consisting of a core of 0.3 g of cow's milk protein/kg/day
plus an amino acid mixture in proportions and amounts equal to 0.9 g milk
protein/kg/day. Diets provided 100 kcal/kg/day with proper vitamin and
mineral supplements. The single essential amino acid under study was
partially replaced in the diet by glycine at five different levels.
Nitrogen balance (4 day periods) was calculated with an allowance of
8 mg N/kg/day for integumental losses. It was assumed that a retention of
16 mg N/kg/day allowed for normal growth and results were validated with
studies conducted in children fed milk or soy protein to assess protein
needs. The following requirements were suggested as mg/kg/day: isoleucine,
31; lysine, 64; sulfur amino acids, 27; threonine, 37; and tryptophan, 14.
The investigators concluded that for children in this age group the FAO/WHO
1973 estimates may be too high for isoleucine, sulfur amino acids, threonine
and valine. Fasting plasma amino acid levels were also measured and it
was concluded that reductions in specific amino acids levels occur in
plasma as amino acid intakes are decreased to sub-optimal levels. Thus,
in young children as in infants, fasting plasma amino acid levels appear
useful in estimating amino acid requirements.

Graham, MacLean and co-workers have data in both infants and young
children fed soy protein (5) or wheat protein (6), that the amino acid
known to be limiting in these proteins is decreased in plasma 3 hrs post-prandial
in contrast to other amino acids, which are either increased or
stable. Supplementation with methionine for soy and lysine for wheat
corrected this condition. Thus the posprandial plasma amino acid response
seems to be helpful in quantitating amino acid needs in growing children.
Fomon (7) has also shown that in infants fed a soy bean diet when a
supplement of methionine was added, weight gain improved and the serum
urea nitrogen level decreased. Hence for infants and young children
both amino acid and urea concentrations in plasma might be employed to
gain information on amino acid requirements.

Amino Acid Requirements of Adults

Evidence is accumulating that histidine is an essential amino acid
for adults. It was found that when uremic patients receiving mixtures
of the Rose eight essential amino acids were given a histidine supplement,
hemoglobin increased (8) and nitrogen retention improved (9). Later
Kopple et al. (10) studied 3 normal (ages 48, 38 and 24 yrs) and 3 uremic
men (ages 55, 54, 43 yrs) who ingested a histidine-deficient amino acid
diet of approximately 6.5 g N/day. These subjects except for one individual
went into negative nitrogen balance after periods of 20 to 30 days. Strong
positive nitrogen balance was immediately restored on the addition of
1200 mg of dietary histidine in all but the one subject mentioned previously
who was in negative balance after 5 days of receiving the histidine-deficient
diet. In 24 hrs plasma histidine levels fell to 52% of control
values with the 40 g protein diet. By the end of the histidine-depletion
period, plasma histidine levels were 17% of control values. Muscle free
histidine concentrations also decreased. Serum albumin and hematocrits
fell and serum iron rose. Subjects felt unwell and in five subjects a
erythematous skin lesion appeared. All clinical symptoms and skin lesions
disappeared with the histidine supplement. Anderson et al. (11) have also
investigated six college-age men consuming for 1 week 6.3 g N amino acid
diets with or without histidine or histidine plus arginine. Mean nitrogen
balances (last 4 days of period) were positive only when histidine was
present in the diet. Cho et al. (12) observed reduced plasma histidine
levels when the young men referred to above ingested low-histidine diets.
In further studies of the amounts of histidine that may be required to
maintain nitrogen balance over prolonged time periods, 9 normal and 4
uremic men were fed amino acid diets containing 6.5 g N and 4, 8, or 12 mg/kg
of histidine for 27 ± 5 days per period (13). The average age of subjects
was 44 years. Fasting plasma histidine levels rose with increasing histidine
intake and 1 hr postprandial levels were higher only in subjects ingesting
12 mg histidine/kg. Likewise the uptake of radio-iron into red cells was
lower than the normal range for all subjects ingesting 4 mg histidine/kg,
lower in 3 of 6 subjects receiving 8 mg/kg and within the range for 5
subjects fed 12 mg/kg. From these data, it appears that the dietary
histidine requirement in both normal and uremic men is more than 8 mg/kg/day
and may be as high as 12 mg/kg/day. For a 70 kg man the range is from
560 to 840 mg of histidine per day.

These observations indicate that histidine is unique among essential
amino acids in that short-term nitrogen equilibrium is maintained with
extremely low histidine-containing diets. There are several hypotheses to
explain this phenomenon: 1) histidine comprises eight percent of the
hemoglobin molecule and the breakdown of hemoglobin contributes more
histidine in proportion to other essential amino acids; 2) the hydrolysis
of the dipeptide carnosine from muscle releases histidine. There is good
evidence that in rats and chicks the muscle carnosine content is decreased
with histidine-free diet and replenished with histidine supplementation
(14, 15) and 3) there is some biosynthesis of histidine (16). A combination
of these hypotheses may provide the explanation for the unique response of
body tissues to the dietary removal of histidine. No reports of histidine
requirements of women have been found. However, it is possible that in
women fed histidine-deficient diets negative nitrogen balance might develop
more rapidly than in men because of a decreased muscle mass and thus
perhaps more limited carnosine stores.

Young and co-workers have presented additional data on valine, lysine
(17), tryptophan (18, 19) and threonine requirements (20) using nitrogen
balance and the plasma amino acid response curve. In human subjects,
for some amino acids at least, as dietary intake is increased, plasma
levels remain stable for a time, then rise abruptly to be followed by
another plateau. Young has attempted to quantitate the amino acid intake
at the point where plasma levels begin to increase, the “breakpoint” and
compare this intake level with the amount needed to maintain nitrogen
equilibrium. These investigators have also used postprandial amino acid
levels as indicators of dietary requirements. They have found that in
general the lower “breakpoint” of the plasma amino acid response curve
corresponds to the minimum levels of amino acid intake that is necessary to
maintain nitrogen balance. It is possible therefore in the case of
essential amino acids with plasma levels responsive to dietary intake
that criteria based on plasma amino acid concentrations can be developed
to assess requirements.

The investigations of Young and co-workers have included elderly
subjects and have provided new data on specific essential amino acid
requirements for subjects in this age group. When subjects were ingesting
0.5 g protein/kg body wt, the mean minimum requirement of elderly people
for tryptophan was 2 mg/kg body weight/day and for threonine it was 8 mg/kg.
These values are similar to those estimated for young subjects.

Plasma response curves have also been used in an attempt to evaluate
histidine requirements (21). Five normal and two uremic men received
amino acid diets in which histidine intakes were varied from 60 to 2800 mg/
day at 8-day intervals. Postabsorption plasma and urinary histidine levels
were correlated with histidine intake but the plasma response curve did
not demonstrate a consistent breakpoint which could be used to indicate
histidine requirement. When 6 normal subjects were fed 2 mg/kg/day or
less, nitrogen balance (not corrected, for integumental losses) was
-0.18 ± 0.5 mg/kg/day, suggesting that histidine requirements are greater
than this amount.

Summary of Current Evidence on Amino Acid Requirements:

The conclusions of the reviewers of amino acid requirement studies
in 1971 (1) appear to be quite relevant even today. Data are still
lacking in children of some age groups and in women during pregnancy and
lactation. The number of adult subjects who have been studied in terms
of needs for specific essential amino acids is small and there is an apparent
marked variability among individuals. Also, differences are great between
study designs. The NRC/FN Committee on Amino Acids (22) has indicated
that with present criteria of estimation, it seems unlikely that there is
a difference in essential amino acid requirements expressed per kilogram of
body weight between men and women. This conclusion would appear to be
still valid.

It would appear that although there are some conflicting data, the
preponderance of studies suggests that essential amino acid requirements
of the elderly do not differ from younger age groups, at least when the
total nitrogen intake is low.

Factors to be Considered in Assessing Essential Amino Acid Requirements:

It is becoming increasingly apparent that a knowledge of essential
amino acid requirements is of great practical value for evaluating food
supplies and instituting supplementation when it is needed. It would
appear that a considerable expansion of the amino acid requirement studies
is a necessary prerequisite for obtaining a valid data base and factors
to be considered in designing future investigations should be carefully
evaluated.

Energy intake: Recent studies of protein requirements have clarified
the quantitative relationships between total nitrogen and energy intake
(23, 24). Calloway (24) has found that 68 ± 15 mg/kg of egg N maintained
nitrogen balance and weight in young men with a dietary intake of 43 ±
4.4 kcal/kg but with an intake of 39.6 ± 4.4 kcal, 89 ± 18 mg N per kg was
required. A slight adjustment in energy intake with marginal N intake
can substantially perturb N balance. It has been often stated that amino
acid requirements are based on dietary energy contents that in many
instances were higher than the amount required to maintain weight and were
above the usual intake. In the ideal study of amino acid needs, weight
and body composition should be maintained.

Length of study period: Amino acid requirements were formulated on
the basis of short-term nitrogen balance studies. However protein allowances
sufficient to promote short-term nitrogen balance have now been found to
be inadequate to maintain long-term nitrogen balance and body potassium
content (25). Although there is evidence that the limiting nitrogen component
in these studies is non-protein nitrogen (26) these results bring
into focus the possibility that the minimum or “safe” amount of one or more
of the essential amino acids also may not be sufficient for prolonged
periods. It is now known that dietary histidine inadequacy affects nitrogen
balance only after a period of 15–20 days or longer (vide infra)(10).

Total nitrogen intake: There is now good evidence, including the
study referred to above (26) that the limiting nitrogen component in
high quality proteins is not the essential but rather the nonessential amino
acids (27, 28). When nonessential nitrogen is added to high quality protein,
nitrogen balance is promoted. There are also other circumstances related
to nonessential nitrogen additions where nitrogen balance could be enhanced.
When energy intake is limited, additional nonessential nitrogen through
provision of extra calories could increase nitrogen retention. It is also
possible that extra nonessential nitrogen may increase requirements for
essential amino acids. In older individuals there is limited evidence
that the requirement for an essential amino acid mixture increases as
the total nitrogen intake is increased from 7 g to 10 g/day and the requirement
lessened as total nitrogen intake is reduced from 7 g to 3.5 g/day (29).
It is also possible that the oxidation rate of certain amino acids may be
affected by protein intake. Limited evidence with injection of labeled
tyrosine indicates less expired 14CO2 and therefore decreased oxidation
in uremic men fed 20 as compared to 40 or 60 g protein diets (30). Enhanced
oxidation of amino acids could also be a factor in increasing amino acid
requirements with higher protein intakes.

Labile body protein: There is firm information that the protein
content of some organs such as liver, pancreas and gut respond rapidly to
changes in protein intake (reviewed by Munro, 31). The amount of nitrogen
lost in the urine during the first few days of ingesting a protein-free
diet is considered to represent the labile protein reserve which accumulates
when dietary protein is high and recedes when protein intake is lowered.
Munro estimates the capacity of the human body to accumulate labile protein
as 300–400 g. Maintaining this labile protein must represent a metabolic
cost of extra protein and extra energy. When subjects are given protein-free
diets prior to test treatments with amino acid mixtures or when subjects
are in negative nitrogen balance for prolonged periods of time, the labile
protein of the body is depleted and as a consequence it can be presumed
that the amino acid requirement would be decreased. In the study by
Young on tryptophan requirements (19), when the dietary tryptophan intake
was 4 mg/kg/day, 6 subjects who had received protein-free diets for 2 days
were in positive nitrogen balance and 8 subjects who had not been given the
protein-free diet were in negative nitrogen balance. In future studies of
amino acid requirements more attention should be given to the state of
body labile protein stores. In addition to the amount of nonessential
nitrogen supplied to maintain these stores it is possible the kind of non-essential
nitrogen has some influence (32). The limited evidence that
older individuals might have increased essential amino acid requirements (30, 33)
was obtained only in studies with subjects receiving diets of 7 or 15 g of
nitrogen. Since each diet trial period was interspersed with an isonitrogenous
period of ordinary food, labile body protein stores would appear to have
been well-maintained.

Methods of assessment: The limitations of the nitrogen balance method
have been reviewed many times and the recent results of long-term studies
have made it clear that this method cannot be the sole criterion for
estimating amino acid requirements in adult subjects. Thus other methods
must be introduced and validated to accurately assess essential amino acid
needs. There is quite good evidence that in infants and children plasma
amino acid levels are responsive to dietary intake and can indicate amino
acid deficiencies. Graham and co-workers (5, 6) are actively exploring
the usefulness of both fasting and postprandial amino acid levels in
relation to estimating limiting amino acid needs when various vegetable
proteins are fed. In adult subjects, Young and co-workers are investigating
the plasma acid response curve as described above (17, 18, 19, 20) and also
postprandial amino acid levels as indicators of amino acid needs. These
would appear to be methods of promise for evaluating amino acid requirements.

In the development of new approaches for evaluating essential amino
acid requirements, perhaps there can be methods appropriate for only a
single amino acid which can be based on a specific metabolic need for this
amino acid.

The usefulness of alterations in some of the blood proteins with
short half lives such as transferrin and retinol binding protein should be
explored. The sophisticated studies of Young and Bier and co-workers using
infusions of stable isotope-labeled amino acids to measure turnover may
provide the ultimate approach to assessing amino acid requirements with
different dietary conditions (34, 35).

Requirement for histidine: It has been established that short-term
nitrogen balance studies are not useful in assessing daily histidine
needs (10, 11). The daily requirement estimates of 8 to 12 mg/kg are
based on results obtained from labeled iron uptake into red cells and the
data have not been published in detail. The true histidine requirement
would appear to consist of maintaining not only body protein but also
carnosine stores. The response curve of plasma histidine to dietary
histidine did not demonstrate a consistent breakpoint that could be useful
in indicating the histidine requirement (21). However both postabsorptive
plasma histidine and urinary histidine levels are correlated with dietary
histidine intake (21) and it would appear that these parameters might
ultimately prove useful in evaluating histidine requirements.